1. Field of the Invention
The present invention relates to a fluorescence spectrophotometer and a fluorescence spectrophotometry and imaging method.
2. Description of the Related Art
There has been known a fluorescence spectrophotometer, which is configured to analyze a sealed material or liquid enclosed (contained) in a sample container (“Fluorometry—Applications to Biological Science”, The Spectroscopical Society of Japan —Measurement Method Series 3, pp. 45-77, Japan Scientific Societies Press, Jan. 20, 1983 (hereinafter referred to as “Non-patent Document 1”)). There has also been known a fluorescence spectrophotometer, which uses an integrating sphere to correct a fluorescence spectrum (“A Simple Correction Method for Determination of Absolute Fluorescence Quantum Yields of Solid Samples with a Conventional Fluorescence Spectrophotometer”, Japan analyst, Volume 58, Issue 6, pp. 553-559, 2009 (hereinafter referred to as “Non-patent Document 2”)). There has further been known a fluorescence fingerprint imaging device to which a technology of spectral imaging for acquiring a spectral image is applied (“Development of the Fluorescence Fingerprint Imaging Technique”, Journal of the Japanese Society for Food Science and Technology, Volume 62, Issue 10, 2015 (hereinafter referred to as “Non-patent Document 3”)).
There has been known a fluorescence detection device configured to detect fluorescence of a sample by using, as a light source, an LED that emits single-wavelength excitation light (WO 2017/056830 A1 (hereinafter referred to as “Patent Document 1”)). There has also been known a device configured to acquire fluorescence spectrum information in each pixel of an image by using a hyperspectral camera (Japanese Patent Application Laid-open No. 2013-137199 (hereinafter referred to as “Patent Document 2”)).
Such a fluorescence photometer as described in Non-patent Documents 1 and 2 acquires, as an excitation spectrum, a fluorescence spectrum obtained when a sample is placed and irradiated with excitation light or a fluorescence intensity obtained when a wavelength of the excitation light is changed. At this time, a sample chamber is required to be a dark room, and hence an emission distribution, an emission color, an emission intensity, and other such states of fluorescence of the sample at the time of being irradiated with the excitation light cannot be identified.
Meanwhile, such a fluorescence fingerprint imaging device as described in Non-patent Document 3 acquires emission information of fluorescence as an image of an in-plane distribution. With this method, fluorescence information obtained when the sample is irradiated with freely-selected excitation light can be acquired as the image. At this time, white light is separated by optical filters, and hence an excitation wavelength is limited by the number of optical filters. Moreover, the sample is directly irradiated with the excitation light through a lens, and hence an excitation light irradiation density is biased toward a center portion, with the result that an amount of excitation light becomes uneven. The amount of excitation light and the fluorescence intensity are in a proportional relationship, and hence the unevenness in amount of light may lead to an uneven fluorescence image in the plane of the sample.
In Patent Document 1, as in Non-patent Document 3, there is acquired image data focusing attention on fluorescence generated under the state in which the sample is irradiated with the excitation light. In Patent Document 1, an LED having a single emission wavelength is used as the light source of the excitation light. A plurality of LEDs as the light sources of the excitation light may be prepared depending on samples to excite the samples with different excitation wavelengths, but the excitation wavelengths are limited to the number of LEDs. Moreover, in Non-patent Document and Patent Document 1, the emission information of the fluorescence is acquired as the image of the in-plane distribution. However, a fluorescence spectrum cannot be acquired, and an intensity of the fluorescence is merely grasped in the image.
With the device described in Patent Document 2, the fluorescence spectrum information in each pixel in the image can be acquired with the use of the hyperspectral camera. However, the hyperspectral camera is expensive, and a data acquisition interval of the spectrum has a resolution that is as low as about 5 nm. Moreover, the sample is directly irradiated with the excitation light, with the result that the amount of excitation light becomes uneven, and hence a sample image in the plane of the sample may become uneven.
Moreover, as described in Non-patent Document 3, in a related-art measurement site, it is common to acquire spectral information with the fluorescence photometer, and narrow down excitation and fluorescence wavelength conditions based on the information to acquire a sample image of fluorescence. Therefore, it is a common method to display information of the spectrum and the sample image separately and independently.
In other words, it is common to acquire the information of the spectrum and the sample image with different devices, and to display the information separately. The spectrum is, so to speak, average information obtained by averaging the entire region irradiated with light, and for a sample having uneven properties for each region, it has been difficult to identify such unevenness. Meanwhile, information on such unevenness can be obtained from the sample image, but it has been difficult to check the information along with the spectrum indicating the overall properties. It is desired to measure the sample taking in consideration both the spectrum indicating the overall properties of the sample and the sample image indicating the properties of each region of the sample. It is true not only for the fluorescence photometer but also for other photometric analyzers.